Climate‐driven mitochondrial selection in lacertid lizards

Abstract The mitochondrion, which is an intracellular organelle responsible for most of the energy‐producing pathways, can have its genome targeted for climate‐driven selection. However, climate‐driven mitochondrial selection remains a sparsely studied area in reptiles. Here, we reported the complete mitochondrial genome sequence of a lacertid lizard (Takydromus intermedius) and used mitogenomes from 54 species of lacertid lizards to study their phylogenetic relationships and to identify the mitochondrial genes under positive selection by climate. The length of the complete mitochondrial genome sequence of T. intermedius was 17,713 bp, which was within the range of lengths (17,224–18,943) ever reported for Takydromus species. The arrangement of mitochondrial genes in T. intermedius was the same as in other congeneric species. The 54 lacertid species could be divided into three geographically and climatically different clades. We identified three mitochondrial genes (ATP6, ATP8, and ND3) under positive selection by climate, and found that isothermality, temperature seasonality, precipitation of wettest month, and precipitation seasonality were the most important climatic variables contributing to the gene selection.

have been extensively used to infer phylogenetic relationships and evolutionary processes.The mitogenome is typically a closed circular double-stranded DNA molecule, often 15-20 kb in length and coding 37 genes, including 22 transfer RNA (tRNA) genes, 13 proteincoding genes (PCGs), and two ribosomal RNA (rRNA) genes; it also includes a noncoding (control) region of variable length that plays a regulatory role in transcription and replication (Boore, 1999).The 13 proteins encoded by mitochondrial PCGs are essential components of the enzymes involved in the citric acid cycle and oxidative phosphorylation (OXPHOS) pathway (Ballard & Pichaud, 2014;Chong & Mueller, 2013;Zhang et al., 2021).Mitochondrial genes may experience different selection pressures among populations or species that differ in distribution or use different habitats.For instance, variation in amino acids at certain specific sites of cytochrome c oxidase (COX) proteins encoded by mitochondrial genes may enhance mitochondrial respiration during hypoxia, and is therefore considered to be related to high-altitude adaptation in animals (Luo et al., 2008;Xu et al., 2005).In humans, ATP6, Cytb, COX1, and ND4 have experienced natural selection by climate (Balloux et al., 2009;Mishmar et al., 2003;Zhang et al., 2013).Climate induces the diversifying selection on ND4 and ATP6 in two Tetranychus species of spider mites (Sun et al., 2018).Climate drives the gene selection of ND5, ND6, and COX1 in Australian songbirds (Lamb et al., 2018).Earlier studies consistently suggest that mitochondrial genes are susceptible to climate change.
Lacertidae is not a very species-rich family, currently including 44 genera and some 370 species (http:// repti le-datab ase.repta rium.cz/ ; Arnold et al., 2007).The family has been divided into two subfamilies, Gallotiinar and Lacertinae, with the latter subfamily further divided into two clades (Arnold et al., 2007;Pyron et al., 2013).Phylogenetic relationships within the family Lacertidae were once unclear because of the indistinguishable morphological characteristics such as the relationship between the genera Takydromus and Platyplacopus (Arnold, 1997).The phylogenetic analyses of the mitochondrial DNA (mtDNA) sequences have provided new insight into the evolutionary relationships within Lacertidae (Arnold et al., 2007;Chen et al., 2009;Guo et al., 2011).
For example, the phylogeny inferred from cyt b gene supports the idea that the genus Platyplacopus should be merged into the genus Takydromus (Chen et al., 2009).
Lacertid lizards are widely distributed in Eurasia and Africa (Arnold et al., 2007), covering a range from temperate to tropical areas with a wide variety of climatic conditions that may induce geographic variation in metabolic rates and thermoregulation ability (Guo, Zhong, Xie, et al., 2021;Guo, Zhong, Zhu, et al., 2021;He et al., 2013).However, whether climatic differences may lead to differential selection pressures on the mtDNA in the family Lacertidae remains unknown.Here, we reported the complete mitochondrial genome sequence of a lacertid lizard (Takydromus intermedius) and used mitogenoms from 54 lacertid species to study their phylogenetic relationships and to identify the mitochondrial genes under positive selection by climate, aiming to investigate climate-driven mitochondrial selection in lizards.

| Sample collection and DNA extraction
We collected an adult female of T. intermedius from Bashu (106°55′ N, 29°10′ E), Chongqing in July 2021.The lizard was brought to our laboratory at Wenzhou University, where the most distant 15 mm of its tail tip was excised using a sterilized scalpel.The lizard was allowed to heal the wound for 3 days after the tail-excising event, and was thereafter kept under the laboratory conditions designed specifically for Takydromus lizards (Guo, Zhong, Xie, et al., 2021;Guo, Zhong, Zhu, et al., 2021;Ji et al., 2007;Lin & Ji, 2005;Luo et al., 2010;Ma et al., 2019).The lizard died 8 months after it was collected.We preserved its body in 95% ethanol and stored it in our laboratory for possible use later.We used the DNeasy Tissue Kit (Qiagen, Germany) to extract total genomic DNA from muscle tissue of the tail sample, following the manufacturer's instructions.

| Mitochondrial DNA amplification and sequence analysis
We designed 15 pairs of primers (Table S1) to amplify contiguous and overlapping fragments of the complete mitochondrial genome, following the procedures described in earlier studies of other Takydromus species.The amplifications were performed with Taq DNA polymerase (TaKaRa, Dalian, China) in a PCR Instrument (Biometra Tone 96, Jena, Germany).The products were sequenced by Sangon Biotech (Shanghai) Co., Ltd.The PCR process and sequencing were repeated twice to ensure the accuracy of the results.
The mitogenome sequence was manually aligned and corrected using the Conting Express 9.1.We used the MITOS (http:// mitos.bioinf.uni-leipz ig.de/ index.py) online server (Kumar et al., 2016;Lamb et al., 2018) to locate the position of each of 37 genes (13 proteincoding genes, two rRNA genes and 22 tRNA genes).The MITOS was also used to identify the tRNA secondary structures and positions according to that in other lizards.The start and end codons of all 13 PCGs were tested in Open Reading Frame Finder via NCBI (https:// www.ncbi.nlm.nih.gov/ orffi nder/ ) and MITOS based on the annotated mitogenome of T. amurensis.

| Phylogenetic analysis
We downloaded the mitogenome sequences of other 53 lacertid species from the NCBI database to analyze phylogenetic relationships within Lacertidae (Table S2).We used MrBayes 3.2.6 under GTR + I + G + F (50,000,000 generations) with the initial 25% burn-in (Ronquist et al., 2012) to construct the Bayesian Inference (BI) tree for the 56 species, including 54 lacertid species and two gekkonid species of the genus Gekko (G.subpalmatus and G. hokounesis), of which the latter two were used as outgroups (Table S2).The nucleotide sequences of 13 PCGs and two rRNAs (12S rRNA and 16S rRNA) were used in phylogenetic analysis and aligned by MUSCLE codon alignment implemented in MEGA 7 (Kumar et al., 2016).

| Climatic variables
We used the point sampling tool in DIVA GIS to extract values for each climatic variable of the distribution points from the 54 lacertid species with a spatial resolution of 30 arc seconds (~1 km) from raster layers in WorldClim 1.4.(www.world clim.org/ current).Nineteen climatic variables were used to represent the climatic drivers of selection (Guo, Zhong, Xie, et al., 2021;Guo, Zhong, Zhu, et al., 2021).According to the climatic variables >50% distribution areas, we assigned the habitats of the 54 species of lacertid lizards into one of three (tropic, subtropic, and temperate) groups.The 19 climatic variables were standardized within the range from −1 to 1 before they were used in a principal components analysis (PCA) for climatic classification of the habitats.We used R 3.6.1 to perform phylogenetic generalized least squares (PGLS) regressions in Caper 1.0.1 package (Orme et al., 2018) and thereby tested whether climatic classification of the habitats was consistent with phylogenetic relationships within Lacertidae.

| Molecular evolution analyses
We used phylogenetic analysis by maximum likelihood in EasyCodeML to estimate the ratio of nonsynonymous to synonymous substitution ratios (ω = dN/dS) and to analyze the episodic gene selection on particular sites in mitogenomes in a climatic category (Gao et al., 2019;Weadick & Chang, 2012;Yang, 2007).
The Clade model is usually used to accommodate site-specific divergence in selective constraint for two or more clades in the tree (Forsberg & Christiansen, 2003).We chose the most commonly used Clade model C (CmC) to estimate separate ω ratios along particular branches (Weadick & Chang, 2012).Based on the topology analysis of the 54 lacertid lizards, we used Clade model to evaluate the evolutionary rate for each branch, assigning T. intermedius as foreground and the others as background branches.The commonly used paired models of CmC versus M2a-rel (a model assuming under the same selection pressure) were used to test the significance of the selection in each climate extreme by likelihood ratio tests.
Using the R package gradientForest, we performed gradient forest analysis to detect the differences in positive selection effects of different climate factors on different genes (Ellis et al., 2012).To perform this analysis, we calculated genetic distances (p-distances) of the positive selection genes and fitted them to 19 current climatic variables for the 54 lacertid species including T. intermedius.We then calculated Euclidian distances between the positive selection genes and the climatic variables.A greater Euclidian distance suggests a greater influence of a climatic variable on genetic distance.

| RE SULTS
The length of the complete mitochondrial genome sequence of T. intermedius was 17,713 bp (GenBank accession Number OQ632596), which was within the range of lengths (17,943) ever reported for Takydromus species (Table S3).The gene arrangement of 13 PCGs, 22 tRNAs, two rRNAs (12S rRNA and 16S rRNA), and one D-loop region (2330 bp) in T. intermedius was the same as in other congeneric species.Nine genes, including ND6 and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro), were on the light strand, and the others were on the heavy strand.The AT content (59.9%) of the mitogenome was smaller in T. intermedius than in other Takydromus species studied thus far (Table S3).The AT skew was 0.04 in T. intermedius, which was the same as in T. amurensis and higher than the values reported for other Takydromus species; the GC skew was −0.32 in T. intermedius, which was smaller than the values reported for other Takydromus species (Table S3).The 13 PCGs ranged in size from 162 bp (Atp8) to 1827 bp (ND5) and started with ATG (Table S4).
The 54 lacertid species could be divided into three main clades (Figure 1).More specifically, Gallotia atlantica and Psammodromus algirus formed a clade, Algyroides nigropunctatus, Phoenicolacerta kulzeri, Zootoca vivipara, and species of the genera Darevskia, Lacerta, Podarcis, and Takydromus formed a clade, and Australolacerta australis, Mesalina olivieri, Meroles squamulosus, and species of the genera Acanthodactylus and Eremias formed a clade (Figure 1).Takydromus intermedius was a sister taxon to T. sylvaticus (Figure 1).PCA resolved two components (eigenvalues ≥1) from the 19 climatic variables for the 54 lacertid species, generally confirming the grouping of three climatic types (Figure 2a).PC1 and PC2 accounted for 41.8% and 29.8% of total variance in the original climatic data, respectively (Figure 2a).Acanthodactylus guineensis and T. sexlineatus could be assigned to the tropic group, 18 species including five Takydromus species could be assigned to the subtropic group, and the remaining 32 species mainly including Eremias and Darevskia species could be assigned to the temperate group (Figure 2a).The climatic factors of PC1 and PC2 exhibited strongest phylogenetic signal (λ = 0.869) (Figure 2b).The CodeML CMC revealed five genes (ND2, ATP6, ATP8, ND3, and ND4L) as candidates for positive selection (ω > 1), with three (ATP6, ATP8, and ND3) confirmed by CodeML M2a-rel (Likehood Ratio Test (LRT) p values <.01) (Table 1).Gradient forest analysis revealed that all three positive selection genes were affected by climatic variables, with ATP6 being the most climatically sensitive gene (Figure 3).The top two explanatory climatic variables tested in gradient forest analysis were Bio3 (isothermality) and Bio4 (temperature seasonality).Two precipitation-related variables, Bio13 (precipitation of wettest month) and Bio15 (precipitation seasonality), contributed significantly to variation in the three positively selected genes.

| DISCUSS ION
In this study, we tested for climatic correlates of mitochondrial DNA variation in lacertid lizards.We constructed phylogenetic relationships within Lacertidae, which were generally consistent with the pattern reported by Pyron et al. (2013).Our study is the first to demonstrate that three mitochondrial genes (ATP6, ATP8, and ND3) are under positive selection by climate in lacertid lizards.
More specifically, two temperature-related (Bio3 and Bio4) and two precipitation-related (Bio13 and Bio15) climatic variables contributed significantly to variation in these three positive selection genes.
The gene order in the mitogenome always varies in invertebrates (Pereira, 2000;Yamazaki et al., 1997;Zhang et al., 2021).Gene reversal, gene transposition, and tandem duplications with subsequent random gene loss have been proved to be possible causes of changes in the mitochondrial gene order in deep-sea mussels (Zhang et al., 2021).In contrast, the mitochondrial gene order is conserved in vertebrates (Kundu et al., 2018;Tian & Guo, 2022).Our study supports the conclusion drawn in vertebrates, as revealed by the observation that the mitochondrial gene order was almost identical among different species of lacertid lizards, with inter-specific differences reflected only in whether some genes are located on the heavy or light strand.The AT and GC skews are a measure of strand asymmetry, and relate to gene replication, selection, and mutation; the GC skews are always higher than the AT skews (Hassanin, 2006;Wei et al., 2010).Our data confirmed that the GC skews were higher than the AT skews among lacertid species.In addition, the similar GC skews among lacertid species may reflect the similar strand asymmetry in these reptiles.Strand bias is usually higher in Arthropodas than in reptiles (Hassanin, 2006;Kundu et al., 2018;Wei et al., 2010), The Bayesian phylogenetic tree based on the mitochondrial genes (two rRNA genes and 13 PCGs) of 54 species of lacertid lizards.The model used is the MUSCLE codon.Value on each branch corresponds to the posterior probability obtained with the Bayesian inference analysis.indicating that the frequency of gene mutation may be higher in invertebrates than in vertebrates.
Environmental stressors may result in gene mutation.Each population will produce selection signatures to adapt to its indigenous environments, and then the evolution of genes occurs (Fleming et al., 2017;Pedro et al., 2015).Genetic differences are one of the most important indicators for testing species evolutionary relationships.In this study, the phylogenetic relationship of the Lacertidae family based on 15 mitochondrial genes is basically consistent with previous studies, which ensured the accuracy of subsequent research (Arnold et al., 2007;Pyron et al., 2013).Phylogenetic relationships inferred from mtDNA data show a strong correlation with the climate, indicating that the mitochondrial genes are affected by climate change.In agreement with previous studies on a diverse array of animal taxa including humans (Balloux et al., 2009;Lamb et al., 2018;Mishmar et al., 2003;Sun et al., 2018;Zhang et al., 2013), mitochondrial genes can be selected by climate in lizards, as revealed by the observation that three mitochondrial genes (ATP6, ATP8, and ND3) were under positive selection by climate in lacertid lizards.ATP6 is the mtDNA-encoded subunit that is incorporated into OXPHOS complex V, which has a channel for the protons' flow back into the mitochondrial matrix (da Fonseca et al., 2008;Fernandez-Vizarra et al., 2009;Wittig et al., 2010).
ATP8 is a companion protein of ATP6 in the ATP synthase (Mishmar et al., 2003;Neckelmann et al., 1987;Wallace et al., 1987).ND3 is oxidoreductase, also known as subunits of complex I (NADH dehydrogenase) (Mishmar et al., 2003), which plays essential role in cellular energy production and proton transport (Efremov et al., 2010;Ohnishi, 2010).The gene mutation of ATP and ND will affect the efficiency of proton translocation, and balance the generate heat and ATP synthesis (Brand, 2000;Sun et al., 2018;Wallace, 2005).
Therefore, the gene selection of ATP and ND is simultaneous (Slimen et al., 2017;Sun et al., 2018).Similarly, the climate promotes the gene selection of ATP6, ATP8, and ND3 and increases the adaption to different climate in lizards.
F I G U R E 3 Results of the gradient forest analysis, showing the contribution of each climatic variable to the gene selection.